Coordinated hormone secretion from the different endocrine cell types in the islets of Langerhans is critical to glucose homeostasis. Spatial islet organization and cell-cell communication are disrupted in obesity, insulin resistance and diabetes, leading to loss of coordinated hormone regulation. Despite the critical roles of islet organization and cell-cell communication in islet function, drugs that target them as potential therapies to diabetes have not yet been developed. Roundabout (Robo) receptors are cell adhesion proteins expressed in the islet. Robo receptors can link cell-cell contact to cytoskeleton dynamic and vesicle transport in insulin secretion. The expression Robo in the islets of mice and humans are severely diminished in obesity and diabetes. We have recently found that deletion of Robo1 and Robo2 in beta cells in mice results in loss of islet organization and impaired glucose tolerance. We further demonstrated that the islet phenotype seen upon beta cell-selective deletion of Robo is not due to transdifferentiation of beta cells to alpha or delta cells, nor is it the result of loss of beta cell maturation or beta cell death. Rather, our results led us to hypothesize that expression of Robo in beta cells facilitates correct islet organization which is required for endocrine cell-cell communication and correct glucose response. According to this hypothesis, the downregulation of Robo in obesity disrupts islet organization, leading to loss of cell-cell communication, thus contributing to dysfunctional glucose response. Understanding how expression of Robo in beta cells regulates islet organization and hormone secretion will provide the basis for new pharmacological approaches to diabetes. We will test the above hypothesis with two specific aims: in Aim 1, we will test the hypothesis that deletion of Robo in beta cells impairs insulin and glucagon secretion. We will further test the hypothesis that deletion of Robo disrupts functional beta cell-beta cell coupling using intravital visualization of synchronized insulin secretion. We will also test the hypothesis that Robo regulates hormone secretion through mediating endocrine cell-cell adhesion. In Aim 2, we will determine how expression of Robo in beta cells controls islet organization, and determine the involvement of the Robo ligand, Slit, in this process. We will further identify signaling pathways and downstream Robo targets in beta cells which could account for the defects in islet organization and glucose tolerance. These data will have important impact on diabetes in three translational aspects: 1) Robo signaling may be manipulated to prevent and restore the deterioration in islet organization and endocrine cell-cell communication in type-2 diabetics; 2) Robo signaling may be used to confer correct 3D organization and cell-cell contact in human pluripotent stem cells-derived islet-like clusters in vitro, to generate an unlimited source of bona fide islets for transplantation, and 3) Robo receptors and their extracellular ligand, Slit, may be used in bioengineering approaches for developing matrix scaffolds that could support structural integrity, survival and function in cadaver islets in clinical islet transplantation settings.